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The present invention is directed to novel microtiter-like plates having a patterned arrangement of relatively hydrophilic domains within a relatively hydrophobic field that can be used in improved methods of high throughput screening of biological materials. The invention covers the plates and their uses for dispensing and moving fluids and for running high throughput screens. Also claimed are methods for controlling evaporation based on cooling the plates to the dew point during the dispense and limiting evaporation during incubation by providing a humidifying buffer in the plate design.
The present invention is also directed to a novel device that can be used to transfer fluids from a spatially ordered array of fluids at a first location to a second location, preserving the spatial relationships among the fluids. The device can be used as a top for the microtiter-like plates having a patterned arrangement of relatively hydrophilic domains within a relatively hydrophobic field. Methods of manufacturing and using the device are also provided.
Current methods of drug discovery often involve assessing the biological activity (i.e., screening) of tens or hundreds of thousands of compounds in order to identify a small number of those compounds having a desired activity. The assays are generally carried out in multi-well tissue culture plates called microtiter plates. Microtiter plates are usually made of plastic, with the wells being formed by indentations in the bottom of the microtiter plate. For screening, commonly used microtiter plates have 96 individual wells, although the trend is to use higher density plates of 384, 864, 1536 , 3456, and even 9600 wells. Current 96 well plates are made in a broad variety of shapes, colors, materials, and sizes, but they all have wells that hold volumes of at least tens of microliters, require individual dispensing of reagents into each well, and require individual washing of each well except in the case of select assays in filter bottom plates. Higher density plates typically have wells that hold lower volumes, but such plates are subject to more limitations in that few such plates are available with filters in the bottom and assay preformance is often compromised. Thus, for plates with more than 384 wells, it is currently not feasible to run biological assays requiring a capture and wash step and moving fluids into and out of the narrow wells of such plates requires very precise pipetting.
In general, it is desirable to utilize microtiter plates having the largest possible number of wells per plate and the smallest possible volume per well, in order to maximize the throughput and minimize the mechanical complexity of high throughput screening operations. In addition, the use of smaller volumes per assay is desirable for a number of reasons: conservation of scarce biological and chemical materials, more efficient use of reagents, ability to run assays on primary cells, ability to develop assays faster due to requiring less reagent purification, fewer plates needed to run a given number of assays and thus fewer handling problems and less storage space needed.
While it is desirable to decrease the size of wells in current microtiter plates, there are problems associated with doing so, including, e.g., difficulty pipetting fluids into confined spaces, inadequate and slow mixing, difficulty effecting separations, rapid evaporation times, and limited signal strength during measurement.
It would be highly desirable to have microtiter plates containing as large a number of wells as possible that hold on the order of 10 nl to 10 xcexcl of assay mixture; that are easy to pipette into; facilitate fluid transfer; minimize mixing time; and allow for easy separations and washing. The present invention provides such plates and methods of their use. Importantly, the design of the plates facilitates fluid transfer, making them also a pseudopipetting device.
Another limitation to the miniaturization of current screening systems is that of evaporation of the reagents during dispensing of the reagents. This problem is generally minimized by pipetting in humidified environments or floating a non-miscible, non-volatile solvent on top of the pipetted component. The humidified environment is difficult to regulate, corrosive to automated equipment, and messy due to condensation. It is difficult to find a truly non-miscible solvent to float on the broad variety of chemicals typically tested in a pharmaceutical screen. The present invention provides two methods for controlling evaporation, one based on pipetting assay components at the dew point that is easy to regulate, non corrosive, clean, and practical and the second based on having a humidifying buffer integral to the plate.
The problem of dispensing an array of small volumes containing compounds of interest in a functional form for screening has been a major obstacle toward miniaturization. In the past, the problem of dispensing small volumes containing compounds of interest into or out of the wells of microtiter plates has been accomplished by use of metal pins that need to be washed after each use (such as on the BioMek 2000 High Density Replicator (HDR) tool, see, e.g., Brandt, 1997, J. Biomolec. Screen. 2:111-116); or by pin replicators (such as the pin replicator made by VandP Scientific, Inc., 1997, J. Biomolec. Screen. 2: 118) or by aspirating a relatively large volume (usually at least 100 nl and generally at least a few xcexcl of solution) with a low volume pipetter such as the Packard piezoelectric pipetter or Cartesian""s solenoid based pipetter. The prior art pin tools or pin replicators are not ideal because they need to be washed (leading to possible contamination and loss of time), work at large volumes, do not have the accuracy needed, or are too expensive. In addition, the prior art pin tools do not act as a reusable lid for the storage of low volume (1-2 xcexcl) compound arrays.
Pipetters, such as those listed above, also have their drawbacks. They are very slow and, like the prior art pins, they also need to be washed, leading to possible contamination and loss of time. The pipetters also require significant dead volumes in the 10s if not 1,000s of nanoliters. In addition, the pipetters, like the prior art pin tools, do not act as lids.
Given the difficulties involved in dispensing and removing reagents or compounds from the wells of microtiter plates, there is a clear need for a device that allows one to remove and then dispense small volumes of reagents from multiple wells simultaneously, without the need to wash the device between uses and without the need to use pipetters with their inherent drawbacks. In particular, such a device that can be activated manually and stabilizes the reagents or compounds for storage would be ideal.
The present invention provides microtiter-like plates containing xe2x80x9cvirtual wells.xe2x80x9d Virtual wells could be any surface modification such as protrusions or slight indentations (e.g., having a depth of between 0.5 nm to 500 xcexcm, preferably about 3 nm to about 200 xcexcm, more preferably about 10 nm to about 100 xcexcm, and even more preferably about 10 nm to about 50 xcexcm), as well as chemical modifications, binding sites, or other discontinuities present in slight indentations, on the plate surface that orders or retains fluid drops into a defined spatial array. Typically, the virtual wells are formed by an arrangement of relatively hydrophilic domains within relatively hydrophobic fields. Solvated samples (compounds) and assay reagents are confined to the more hydrophilic domains of the virtual wells by the edges of the more hydrophobic fields. The use of virtual wells allows one to run high throughput screening assays that require the capture and washing of an assay component prior to reading, as well as assays simply requiring the mixing of components and reading, with assay mixtures having volumes on the order of about 10 nl to 10 xcexcl. The virtual wells can also be used to effect near simultaneous addition or subtraction of fluid from all wells in a virtual well microtiter-like plate to enable screening fast kinetic and flash reactions. The virtual wells allow one to repeatedly transfer a known volume from each well of a spatially defined array of solutions into a second array in Ia single step and to precisely aliquot small volumes of compounds or reagents to multiple assays. The present invention also provides a means for controlling evaporation and providing a reproducible optical path. The present invention also includes methods of high throughput screening utilizing microtiter-like plates containing virtual wells.
The present invention also provides an inexpensive, disposable device for transferring small volumes of an entire array of compounds from a first microtiter-like plate to other microtiter-like plates multiple times, preserving the spatial arrangement of the compounds without risking contamination of samples or requiring excessive washing time. Methods of manufacturing and using the device are also provided.